The present invention relates to recombinant DNA which encodes the BstYI restriction endonuclease (endonuclease) as well as BstYI methyltransferase (methylase), expression of BstYI restriction endonuclease and methylase in E. coli cells containing the recombinant DNA.
BstYI endonuclease is found in the strain of Bacillus stearothermophilus Y406 (New England Biolabs"" strain collection #434). It recognizes the double-stranded DNA sequence 5xe2x80x2Pu/GATCPy3xe2x80x2 and cleaves between the Pu and G to generate a 4-base 5xe2x80x2 overhanging ends (Pu=A or G; Py=T or C;/indicates the cleavage of phosphodiester bond). BstYI methylase (M.BstYI) is also found in the strain of Bacillus stearothermophilus Y406. It recognizes the double-stranded DNA sequence 5xe2x80x2PuGATCPy3xe2x80x2 and modifies the N4-cytosine by addition of a methyl group to become N4-methylcytosine in the DNA sequence. The N4mC modified BstYI site is resistant to BstYI restriction digestion.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2, 5xe2x80x2PuG/GNCCPy3xe2x80x2 and 5xe2x80x2CACNNN/GTG3xe2x80x2 respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2.
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13: 6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535, (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535, 1996). The disadvantage of this method is that sometimes positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2CCWGG3xe2x80x2 (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpM methylase can modify the CG dinucloetide and make the NotI site (5xe2x80x2GCGGCCGC3xe2x80x2) refractory to NotI digestion (New England Biolabs"" Catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the BstYI restriction endonuclease from Bacillus stearothermophilus into E. coli by methylase selection and inverse PCR amplification of the adjacent DNA. A methylase gene with high homology to amino-methyltransferases (N4-cytosine methylases) was found in a DNA library after methylase selection. This gene was named BstYI methylase gene (bstYIM).
In order to clone the BstYI endonuclease gene in a large DNA fragment, partial HindIII genomic DNA fragment libraries were constructed using vectors pBR322 and pUC19. More methylase positive clones were obtained. However, no endonuclease activity was detected in all M.BstYI positive clones.
To ensure there was sufficient DNA coding capacity on both sides of the bstYIM gene, a Southern blot analysis was performed to generate a restriction map near the bstYIM gene. Restriction mapping indicated that ClaI, NsiI, PvuII, and SphI fragments would have DNA large enough to encode the bstYIR gene on either side. Again, methylase positive clones were derived from the new libraries, but no endonuclease activity was detected in cell extract or purified cell extract fractions. More than 14 kb of DNA was derived from one side of the bstYIM. It was extremely difficult to obtain DNA on the other side of bstYIM. The DNA fragment that was difficult to clone simultaneously with bstYIM gene might contain the bstYIR gene.
To screen for DNA damage induced by expression of the BstYI endonuclease or methylation dependent restriction systems, primary plasmid library DNA was used to transform into a dinD::lacZ fusion strain AP1-200 and plated on X-gal plates. A number of blue colonies were found, but none of them contained the BstYI endonuclease gene.
Since both methylase selection and SOS induction assays failed to yield a BstYI endonuclease clone, inverse PCR was employed to amplify the adjacent downstream DNA sequence. An open reading frame was found adjacent to the bstYIM gene. This ORF was named bstYIR and expressed in a T7 expression vector pET21b. This clone produced more than 106 units of BstYI per gram of cells. However, this clone was unstable due to a high expression level under non-induced conditions. To construct a more stable clone, an internal NdeI site was mutagenized and the bstYIR gene (an NdeI-XhoI fragment) was then inserted into a T7 expression vector with 4 copies of a transcription terminator upstream of the T7 promoter.
To overexpress the bstYIM gene, the gene was amplified by PCR and cloned into expression vector TYB2. The M.BstYI was expressed as a fusion to chitin binding domain and intein. The host ER2566 (an E. coli B strain) was found to be intolerant to overexpression of M.BstYI and gave a low yield of M.BstYI protein. The plasmid TYB2-BstYIM was later transferred into E. coli K strain ER1821(xcexDE3), a strain deficient in methylation dependent restriction systems (McrBCxe2x88x92 McrAxe2x88x92, Mrrxe2x88x92). The M.BstYI protein was purified by chromatography through affinity, anion exchange, and cation exchange columns.